X-ray fluorescence (XRF) spectrometry is a powerful spectroscopic technique that has been used to determine the elements that are present in a chemical sample, and to determine the quantity of those elements in the sample. The underlying physical principle of the method is that when an atom of a particular element is irradiated with X-ray radiation, the atom ejects a core electron such as a K shell electron. The resulting atom is then in an excited state, and it can return to the ground state by replacing the ejected electron with an electron from a higher energy orbital. This is accompanied by the emission of a photon. The energy of the emitted photons is equal to the difference in the energies of the two orbitals. Each element has a characteristic set of orbital energies and therefore, a characteristic X-ray fluorescence (XRF) spectrum.
An XRF spectrometer is an apparatus capable of irradiating a sample with an X-ray beam, detecting the X-ray fluorescence from the sample, and using the X-ray fluorescence to determine which elements are present in the sample and measuring the quantity of these elements. A typical, commercially available energy dispersive X-ray fluorescence spectrometer is the EDAX Eagle XPL energy dispersive X-ray fluorescence spectrometer, equipped with a microfocus X-ray tube, lithium drifted silicon solid-state detector, processing electronics, and vendor supplied operating software, available from the EDAX division of Ametek, 91 McKee Drive Mahwah, N.J. 07430. An example of a wavelength dispersive X-ray fluorescence spectrometer is the ZSX Primus, available from Rigaku Americas, 9009 New Trails Drive, The Woodlands, Tex. 77381. In principle, any element may be detected and quantified with XRF.
Typical protein-drug assays are performed with nanomolar to micromolar protein concentrations and drug concentrations. The protein concentration and the drug concentration need not be the same. The existing art for the analysis of dried samples by x-ray fluorescence can only measure dried samples which are deposited from one microliter or less of solutions having minimum concentrations of about 10 micromolar (see Thomasin C. Miller, Christopher M. Sparks, George J. Havrilla, Meredith R. Beebe, Semiconductor applications of nanoliter droplet methodology with total reflection X-ray fluorescence analysis, Spectrochimica Acta Part B 59 (2004) 1117-1124, incorporated herein by reference). This sample preparation method is insufficient for the analysis of proteins and protein-drug complexes, where biologically relevant concentrations of the solution from which the sample is deposited must be less than 10 micromolar and preferably less than 100 nanomolar.
The existing state of the art is insufficient for analyzing dilute solutions, especially dilute solutions of proteins having concentrations of less than about 10 micromolar.
There remains a need for simpler methods for preparing samples for measurement using x-ray fluorescence spectrometry. The present invention is designed to address that need.